International Journal of Food Microbiology 334 (2020) 108799

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Genetic polymorphisms associated to SDHI fungicides resistance in selected T

Aspergillus flavus strains and relation with aflatoxin production
⁎
M. Masiello1, S. Somma1, M. Haidukowski, A.F. Logrieco, A. Moretti
Institute of Sciences of Food Production, Research National Council (ISPA-CNR), Via Amendola 122/O, 70126 Bari, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Aspergillus flavus is a common and ubiquitous fungal species able to colonize several agricultural commodities, in
Boscalid both pre- and post-harvest conditions. This species represents a very harmful plant pathogen for its ability to
Isopyrazam synthesize aflatoxin B1, responsible for human primary hepatocellular carcinoma and classified as a group I
Sdh genes (human carcinogenic) by the International Agency for Research on Cancer. Several approaches have been
Resistant genotypes
proposed to control A. flavus development and related aflatoxin production in field and storage conditions. The
Aflatoxins
A. flavus depigmentation
Succinate Dehydrogenase Inhibitor (SDHI) fungicide boscalid has been shown to control A. flavus growth and
aflatoxin contamination both in vitro and in field experiments. However, this compound is classified as medium-
high risk fungicide for triggering fungal resistance and, indeed, resistant strains can occur on crops treated with
boscalid.
In this paper, we selected laboratory A. flavus strains resistant to boscalid grown on agar medium containing
50 mg/L of boscalid. In order to investigate the molecular mechanism responsible for the resistant phenotype,
specific primer pairs were designed to amplify the whole SdhB, SdhC and SdhD genes. By amino acid sequence
analysis, two point mutations, Tyrosine replacing Histidine at codon 249 of SdhB (H249Y) and Arginine re-
placing Glycine at codon 91 of SdhC (G91R), were identified. The effect of SDHI boscalid and isopyrazam on
mycelial growth and conidial germination was evaluated. Both resistant genotypes showed high resistance (MIC
and EC50 > 1000 mg/L) to boscalid. A positive cross-resistance was found between boscalid and isopyrazam.
Specific sub-lethal doses of both fungicides (0.5 mg/L of boscalid and 0.01 mg/L of isopyrazam) interfered
with the mechanisms associated to pigmentation of colonies. In particular, fungal colonies appeared depig-
mented lacking the typical A. flavus green colour shown on un-amended fungicide medium. A strict correlation
between lack of pigmentation and increasing aflatoxin production was also observed.

1. Introduction 2003; Horn, 2007).

In particular, aflatoxins are the most potent carcinogenic, terato-
Aspergillus flavus is a common and ubiquitous fungal species de- genic and mutagenic natural compounds (IARC, 2002). Indeed AFB1 is
tected worldwide on several important crops and agricultural com- classified as a group 1 human carcinogen by the International Agency
modities, such as maize, rice, peanuts, dried fruits and spices (Kabak for Research on Cancer (IARC), since responsible for human primary
and Dobson, 2017; Katsurayama et al., 2018; Iamanaka et al., 2007; hepatocellular carcinoma (IARC, 1993).
Taniwaki et al., 2018). The fungus is able to colonize agricultural The biosynthesis of AFs involves at least 23 enzymatic reactions,
commodities in both field and storage conditions, causing high concern and 15 intermediates of this pathway have been identified (Yu et al.,
among farmers for the high economic losses. In addition, A. flavus re- 2004). The structural similarity of the pigments of A. flavus conidia with
presents a toxicological risk for its ability to synthesize several myco- norsolorinic acid, the first stable intermediate in the aflatoxin bio-
toxins (Cary et al., 2018), in particular such as aflatoxins (AFs), cy- synthesis, makes it possible to suppose that A. flavus pigments and AFs
clopiazonic acid, kojic acid, and sterigmatocystin, which are dangerous have common precursors (Dzhavakhiya et al., 2016). Furthermore, AFs
for human and animals health (Abbas et al., 2009; Bennett and Klich, and melanin biosynthetic pathways have common initial steps (Brown

⁎
Corresponding author.
E-mail addresses: [email protected] (M. Masiello), [email protected] (S. Somma), [email protected] (M. Haidukowski),
[email protected] (A.F. Logrieco), [email protected] (A. Moretti).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ijfoodmicro.2020.108799
Received 1 April 2020; Received in revised form 17 July 2020; Accepted 24 July 2020
Available online 31 July 2020
0168-1605/ © 2020 Elsevier B.V. All rights reserved.
M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

and Salvo, 1994; Pal et al., 2014). In the last years, some studies de- sub-units, the cytochrome b (SdhC) and the CybS protein (SdhD;
monstrated that compounds which blocked pigments synthesis led to a Hägerhäll, 1997).
simultaneous stimulation of the toxinogenesis (Brown and Salvo, 1994; Molecular characterization of the four genes encoding succinate
Dzhavakhiya et al., 2016; Khomutov et al., 2011). Moreover, some dehydrogenase sub-units showed that mutations in the SdhB, SdhC, and
studies report an important role of oxidative stress at the initial steps of SdhD genes leading to amino acid substitutions are responsible for re-
AFB1 biosynthesis (Amare and Keller, 2014; Jayashree and sistance to SDHI fungicides in many phytopathogenic fungi, as de-
Subramanyam, 2000; Montibus et al., 2013; Roze et al., 2013), al- scribed in A. alternata (Avenot et al., 2008a, 2009), Mycosphaerella
though the mechanisms of this effect and the specific forms of reactive graminicola (Fraaije et al., 2012; Scalliet et al., 2012), B. cinerea (De
oxygen affecting the toxinogenesis remain still unclear (Grintzalis et al., Miccolis Angelini et al., 2014), and Penicillium expansum (Malandrakis
2014). et al., 2017) and in other fungal species listed by FRAC.
As reported by Battilani et al. (2016), in the next decades, climatic The present work aimed to: a) select A. flavus strains resistant to
changes and global warming can lead to a greater spread of this pa- boscalid; b) investigate about the molecular mechanisms associated to
thogen also in other geographical areas, such as Southern and Central resistance in A. flavus; c) evaluate the in vitro response of A. flavus
Europe, where is not currently common. Therefore, solutions to reduce boscalid resistant laboratory mutants to both SDHIs boscalid and iso-
AFB1 exposure must be developed in order to face the growing global pyrazam, d) evaluate the possible implications of resistance mutations
health and economic issue represented by aflatoxins (Rushing and towards aflatoxin production.
Selim, 2019).
The use of fungicides is an essential part of agriculture practices 2. Materials and methods
(Brent and Hollomon, 2007) and chemical management of several plant
diseases remains the main tool to control fungal pathogens in most 2.1. Media and fungicides
important crops. However, control of mycotoxin-producing fungi with
fungicides on some extensive field crops is not widely used, because of Commercial formulations of boscalid [Cantus, water-dispersible
the cost and the difficulty of applying agrochemicals at the right crop granules containing 50% active ingredient (a.i.), BASF Italia] and iso-
stage (Lagogianni and Tsitsigiannis, 2018). Furthermore, fungicide re- pyrazam (Zulu, concentrated suspension containing 125 g L−1 a.i.,
sistance can greatly reduce the efficacy of fungicides, increasing both Syngenta) were suspended in sterile distilled water. Then, fungicides
the cost of pest management and the environmental impact, due to a were added to potato dextrose agar (PDA) or water agar (WA), to test
higher number of sprays required to control fungal diseases (Brent, mycelia growth and conidial germination, respectively, cooled down to
2012). 45–50 °C, after autoclaving. Twelve different concentrations of each
Succinate Dehydrogenase Inhibitors (SDHI) are the new fungicide fungicide were tested (0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500,
compounds with the fastest growth into the market, for their high level 1000 mg/L), for both the assays.
of activity against several fungal pathogens (Sierotzki and Scalliet,
2013). They inhibit fungal respiration by blocking the ubiquinone- 2.2. Fungal strains
binding site of the enzyme succinate-ubiquinone oxido-reductase (SDH,
so-called complex II) in the mitochondrial electron transport chain Three A. flavus strains (ITEM 8095, ITEM 8111, ITEM 8115) from
(Hägerhäll, 1997). The first compound belonging to SDHI was carboxin ITEM fungal collection of the Institute of Sciences of Food Production
(Von Schmeling and Kulka, 1966), used mainly for seed treatment (ISPA), isolated from maize kernels in Northern Italy, never exposed to
against basidiomycete pathogens; however, the first SDHI with a broad- SDHI fungicides, were used as wild type strains to establish their re-
spectrum foliar activity was boscalid (Glättli et al., 2011), introduced sponse to the two SDHI fungicides, boscalid and isopyrazam and to
into the market on 2003. Fungicide Resistance Action Committee obtain boscalid resistant strains. Among these strains, only strain ITEM
(FRAC) has listed eighteen SDHI compounds, belonging to different 8095 was selected, because this strain showed the greatest capability to
chemical groups, provided of an extreme large spectrum of action produce conidia.
against fungal pathogens with the exception of oomycete fungi.
In Masiello et al. (2019), A. flavus showed sensitivity in vitro to SDHI 2.3. Isolation of boscalid-resistant mutants
fungicides boscalid and isopyrazam, and a significative reduction of A.
flavus contamination was observed in field trials after treatment with Seven-day-old colonies of sensitive strain ITEM 8095 were flooded
boscalid, making this compound a good candidate to reduce A. flavus with sterile distilled water containing 0.05% Tween 20, and conidia
contamination in field. However, for their site-specific action, these collected by scraping the media surface gently. The conidia were fil-
fungicides are classified by Fungicide Resistance Action Committee tered through Miracloth (Calbiochem, La Jolla, Canada) to remove
(FRAC) as medium-high risk of resistance fungicides (http://www.frac. mycelial fragments.
info). The boscalid resistant mutant were obtained on PDA amended with
First evidence of possible mutations mediating resistance to car- 50 mg/L of boscalid, considered a discriminable concentration able to
boxin were already reported for Ustilago maydis and Aspergillus nidulans block both mycelial growth and conidial germination of sensitive
in the 1970s. Mutant strains, selected after UV irradiation on fungicide strains. In particular, 10 mL of conidial suspension of ITEM 8095 strain
amended media, showed that mutations in succinate dehydrogenase (5 × 109 spore mL−1) were incorporated in autoclaved PDA, cooled
genes were responsible for resistance (Georgopoulos et al., 1972; down to 45–50 °C, under hood, and amended with 50 mg/L of boscalid,
Georgopoulos and Ziogas, 1977; Van Tuyl, 1975). previously suspended in sterilized water. PDA containing fungicide and
Among the fungicides of this group, resistance to boscalid has been conidial suspension were plated on Petri dishes and incubated at
firstly reported in Alternaria alternata, on pistachio in California (Avenot 25 ± 1 °C. Colonies developing within 10 days were individually
and Michailides, 2007). Therefore, several reports of boscalid-resistant transferred on fresh PDA amended with 50 mg/L of boscalid for further
mutants in several other pathogens, including Botrytis cinerea (De analysis. All the grown colonies were subjected to continuous transfer
Miccolis Angelini et al., 2014), Aspergillus oryzae (Shima et al., 2009), on PDA amended with 50 mg/L of boscalid to confirm the stability of
Zymoseptoria tritici (Fraaije et al., 2012), and Pyrenophora teres (Rehfus the fungicide resistance.
et al., 2016), were published.
In fungi, succinate dehydrogenase complex consists of four sub- 2.4. Molecular characterization of SdhB, SdhC and SdhD genes
units: two hydrophilic sub-units, the flavoprotein (SdhA) and the iron-
sulfur protein (SdhB), and two lipophilic membrane-anchored protein Three A. flavus sensitive strains and 7 A. flavus resistant strains were

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

Table 1
Primer pair sequences for Sdh genes and PCR conditions.
Primer pairs Sequences of forward/reverse primers PCR conditions Amplicon size (bp)

Primer concentration in PCR (nM) PCR parameters Cycles

(n)

SdhB.A1Fw 5′-CAGAACTCAGATGCGAACCA-3′ 150 95 °C 2 min; 30 1384

SdhB.A4Rev 5′-AATAAGCGCATCCAACATCA-3′ 95 °C 30 s;
62 °C 30 s;
72 °C 40 s
SdhC.AfFw 5′-TTCCGAAGCGTTACGAGACT −3′ 300 95 °C 2 min; 35 1096
SdhC.AfRev 5′-TTTGCTGTTCACCCTTTCGT-3′ 95 °C 50 s;
59 °C 50 s;
72 °C 1 min
SdhD.AfFw 5′-AGCGTTGAGTCGCAGAGAAT-3′5′-CCAGCTGGACTGGCTTACTT-3′ 300 95 °C 2 min; 30 994
SdhD.AfRev 95 °C 50 s;
60 °C 50 s;
72 °C 1 min

growth on Potato dextrose broth for 3 days at 25 °C, with shaking at actively growing margins of 3–5 day old colonies cultured on PDA,
150 rpm. Genomic DNA to be used in PCR was extracted and purified were used to inoculate Petri dishes (90 mm in diameter) containing
from powdered lyophilized mycelia (10–15 mg) by using the Wizard PDA or PDA amended with fungicides. The inhibition activity of the
Magnetic DNA Purification System for Food kit (Promega Corporation, fungicide on colony growth was determined by measuring the diameter
Madison, WI), according to the manufacture protocol. Quantity and of developing colonies after 3, 5, 7 and 10 days of incubation at
integrity of DNA were checked at Thermo-Scientific Nanodrop (LabX, 25 ± 1 °C, under an alternating light/darkness cycle of 12 h photo-
Midland, ON, Canada) and by comparison with a standard DNA (1 kb period. The inhibition caused by each fungicide concentration was ex-
DNA Ladder, Fermentas GmbH, St. Leon-Rot, Germany) on 0.8% pressed as percentage value, reporting the difference between the
agarose gel, after electrophoretic separation. maximum level of inhibition with no growth of the fungal colony on the
For each A. flavus strain, the SdhB, SdhC and SdhD genes were medium (100%), and the ratio between diameter of colony growth on
amplified and sequenced for further nucleotide and amino acid se- PDA amended with the fungicide and the diameter of the growth of
quences analyses. colony inoculated on PDA medium as control (value of 0% means the
Based on Aspergillus flavus NRRL 3357 nucleotide and amino acid total lack of inhibition corresponding to full growth of the fungal colony
sequences of the Sdh genes, three specific primer pairs were designed to on the medium).
amplify the SdhB, SdhC and SdhD genes, by using the software package In conidial germination test, conidia were collected by scraping the
Primer3 (Rozen and Skaletsky, 2000). All primer pairs were synthesized surface of 7-day-old colonies, grown on PDA at 25 ± 1 °C under an
by Invitrogen - Thermo Fisher Scientific. Sequences of primers and PCR alternating light/darkness cycle of 12 h photoperiod, with a sterilized
conditions are reported in Table 1. loop, suspended in sterile distilled water containing 0.05% Tween 20,
Polymerase Chain Reaction mixture (15 μL) contained 15 ng of DNA and filtered through Miracloth (Calbiochem, La Jolla, Canada) to re-
template, 0.225 or 0.45 μL of each primer (10 mM), 0.3 μL of dNTPs move mycelia fragments. Aliquots (10 μL) of conidial suspension
(10 mM) and 0.075 μL of Hot Master Taq DNA Polymerase (1 U/μL; 5 (1 × 105 conidia mL−1) were spotted on disks (6 mm diam) of WA
Prime). All PCR products were visualized with UV after electrophoretic medium and WA amended with each fungicides concentrations, then
separation in 1× TAE buffer, on 1.5% agarose gel. placed on sterile microscope slides. The disks were incubated in a moist
The PCR products were purified with the enzymatic mixture Exo/ chamber at 25 ± 1 °C in darkness. After 24 and 48 h, 100 conidia
FastAP (Exonuclease I, FastAP thermosensitive alkaline phosphatase, randomly selected on each of three replicated spots were observed at
Thermo Scientific, Lithuania, Europe) and then sequenced by using Big 125× magnification; germinated conidia were counted. The frequency
Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied of conidia able to germinate on medium amended with fungicide was
Biosystems), according to the manufacture's recommendations. Both calculated compared to the frequency of conidia germination on the un-
strands were purified by filtration through Sephadex G-50 (5%) (Sigma- amended control medium.
Aldrich) and sequenced in the 3730xl DNA Analyzer (Applied For both mycelial growth and conidial germination tests, data ob-
Biosystems, Foster City, CA, USA). The FASTA sequences were obtained tained were used to determine EC50 (effective concentration causing
with BioNumerics software (Applied Maths, Kortrijk, Belgium). 50% reduction of colony growth or conidial germination) and MIC
Sequences alignment and translation to obtain amino acid sequences (minimal inhibitory concentration) values.
were performed by using MEGA7 software ver. 7.0.14 (Kumar et al.,
2016).
2.6. Analysis of aflatoxin production in A. flavus agar plate cultures

2.5. Mycelial growth and conidial germination assays Aspergillus flavus wild type strain ITEM 8095 and A. flavus boscalid
resistant mutants were grown on PDA and PDA amended with boscalid
The response to boscalid and isopyrazam fungicides of all fungal (0.5, 1, 5, 10, 50, 100 mg/L) or isopyrazam (0.005, 0.01, 0.05, 0.1, 0.5,
strains was studied by evaluating both colony growth and conidial 1, 5, 10, 50, 100 mg/L). After 14 days of incubation at 25 °C in dark-
germination inhibition. ness, six agar plugs (1 g) of each fungal colony were cut and collected in
Appropriate volumes of each fungicide suspended in distilled sterile tubes for the chemical analyses.
water were added to PDA or WA, to obtain the 12 concentrations of Aflatoxins B (AFBs) were extracted with 5 mL of a solution me-
active ingredient to be tested, and then cooled down to 45–50 °C. Each thanol/water (80:20, v/v) for 60 min at room temperature by shaking
fungicide concentration was tested in triplicate for each fungal strain. at 250 rpm for 60 min (KS 4000i, IKA Werke GmbH & Co. KG., Staufen,
As control, only PDA or WA were used. Germany). Five hundred microliter of extract was diluted with 500 μL
In mycelial growth assay, mycelium disks (4 mm in diameter) from of water and then were filtered using RC 0.20 μm filters.

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

All solvents (HPLC grade) were purchased from J. T. Baker coding sequences of each gene was obtained. SdhB gene had a length of
(Deventer, The Netherlands). Water was of Milli-Q quality (Millipore, 1032 nucleotides (nt) including three introns of 63, 67 and 65 nt, with a
Bedford, MA, USA). The RC 0.2 μm filters (regenerated cellulose Coding DNA Sequence (CDS) of 837 nt; SdhC gene had a length of
membranes) were obtained from Grace (Deerfield, IL, USA). 794 nt including two introns of 57 and 170 nt (CDS 567 nt); SdhD gene
Mycotoxin stock solutions of AFB1 and AFB2 (10 μg/mL each) were (827 nt) included 4 introns of 100, 88, 60 and 57 nt, with a resulting
prepared by dissolving solid commercial toxins in toluene/acetonitrile CDS of 522 nt.
(9:1, v/v) (HPLC grade). The exact concentration of aflatoxin was de- By comparing the nucleotide and amino acid sequences of resistant
termined according to AOAC Official Method 971.22 (AOAC and wild-type strains included in the analysis, it was possible to identify
International, 2000). A stock solution was prepared by mixing the a single point mutation at codon 249 of SdhB gene (H249Y), in which
simple aflatoxin solution and diluting it with toluene/acetonitrile TAC replaced CAC in the strains M1-8095, M2-8095, M4-8095, M5-
(9:1 v/v) into amber silanized vials to obtain a solution containing 8095, M7-8095, and a single point mutation at codon 91 (G91R) of
1 μg/mL of AFB1 and 0.2 μg/mL of AFB2. Aliquots of the stock solution SdhC gene, in which AGA replaced GGA, in the strains M3-8095 and
were transferred to 4 mL amber silanized glass vials and evaporated to M6-8095.
dryness under a stream of nitrogen at 50 °C. The residue was dissolved Nucleotide sequences of A. flavus resistant and wild-type strains
with water/methanol (60:40, v/v) to obtain calibrant standard solu- have been submitted to GeneBank database and can be found under
tions at 0.4, 1.2, 2.0, 4.0, 5.0, 10.0 ng/mL of aflatoxin B1 and 008, 0.24, accession numbers from MT226802 to MT226811 for sdhB gene, from
0.4, 0.8, 1.0 and 2.0 ng/mL of aflatoxin B2. Standard solutions were MT226812 to MT226816 and from MT233443 to MT233445 for sdhC
stored at −20 °C and warmed to room temperature before use. gene and from MT226817 to MT226824 for sdhD gene.
Analyses of AFB1 and AFB2 mycotoxins were performed according
to a UPLC method. The UPLC apparatus consisted of a Waters Acquity 3.2. In vitro response to boscalid and isopyrazam
UPLC system (Milford, MA, USA). Data acquisition and instrument
control were performed by Empower 2 software (Waters). The column Three A. flavus wild-type strains, and 7 boscalid-resistant mutants
used was a 100 mm × 2.1 mm i.d., 1.7 μm, Acquity UPLC® BEH RP-18, were tested for their response to boscalid and isopyrazam, by testing
with an Acquity UPLC column in-line filter (0.2 μm). 7.5 μL volume was both colony growth and conidial germination inhibition.
injected into the UPLC apparatus. The fluorometric detector was set at For each genotype identified by molecular characterization of Sdh
wavelengths of 365 nm (excitation) and 435 nm (emission). The mobile genes, (Wild-Type, H249Y and G91R), dose-response curves for both
phase consisted of a mixture of water/acetonitrile/methanol (64:18:18, colony growth and conidial germination are reported in Figs. 1 and 2.
v/v/v) at a flow rate of 0.4 mL/min. The temperature of the column
was maintained at 40 °C. The AFBs were quantified by measuring peak 3.2.1. Colony growth inhibition
areas at the retention time of aflatoxin standard solutions (Sigma- The mycelial growth of the wild-type sensitive strains ITEM 8095,
Aldrich, Milan, Italy) and comparing these areas with the relevant ca- ITEM 8111, and ITEM 8115 was totally inhibited up to 10 days of in-
libration curve. With this mobile phase, the retention time of AFB2 was cubation by 50 mg/L of boscalid, with EC50 values included in the range
about 2.6 min and AFB1 was about 3.7 min. The LOQ of the method 0.1–0.5 mg/L for the strain ITEM 8095, and 0.05–0.1 mg/L for the
was 1.0 μg/kg for AFB1 and 5.0 μg/kg for AFB2 based on a signal to strains ITEM 8111 and ITEM 8115 (Table 2).
noise ratio of 3:1. All 7 boscalid resistant mutants were able to grow on the highest
fungicide concentration (1000 mg/L) already after 3 days of incubation
2.7. Statistical analyses showing high resistance to boscalid, with MIC and EC50 values higher
than 1000 mg/L after 7 days (Table 2). In particular, as shown in
Statistical analyses of AFBs production at different concentrations of Fig. 1a, at the highest boscalid concentration (1000 mg/L), the strains
both boscalid and isopyrazam were carried out by using the STATIST- M3-8095 and M6-8095 carrying the G91R amino-acid change in the
ICA v. 6.0 software (Stat-Soft, Tulsa, OK). The mean of AFBs production SdhC gene were slightly more inhibited (mean inhibition value 38%)
of all the laboratory mutant strains was calculated for each fungicide than the strains M1-8095, M2-8095, M4-8095, M5-8095, M7-8095,
concentration. The means were then compared by using Tukey's characterized by the H249Y amino-acid change in the SdhB gene (mean
Honestly Significant Difference (HSD) Test, with significance level (P) inhibition value 25%).
of 0.01. During the test to evaluate the mycelial growth inhibition, differ-
The Pearson Correlation values (r) between AFs production and ences in colony colour were observed, up to 10 days of incubation. In
colonies pigmentation of A. flavus strains were calculated for both particular, a strong depigmentation of all the boscalid resistant strain
boscalid and isopyrazam. colonies was observed only when grown on PDA amended with 0.5 and
100 mg/L of boscalid (Fig. 3). The same colonies showed the A. flavus
3. Results typical green colour on PDA and on PDA amended with the other
concentrations of boscalid. When the yellow colonies were transferred
3.1. Molecular characterization of Sdh genes on PDA, they acquired again the green pigmentation, but showed de-
pigmentation if re-cultured on PDA amended with 0.5 and 100 mg/L of
The nucleotide sequences of the three SdhB, SdhC and SdhD subunits boscalid.
were obtained by direct sequencing of PCR amplification products of All the A. flavus strains were also tested on PDA amended with
the three wild type strains and the 7 boscalid resistant strains. isopyrazam (Fig. 2a). The three sensitive strains ITEM 8095, ITEM 8111
For each gene, specific primer pairs were designed on the flanking and ITEM 8115 were totally inhibited by 50 mg/L of isopyrazam, with
regions of SdhB, SdhC and SdhD sequences (NW_002477250, EC50 value included in the range 0.1–0.5 mg/L (Table 2). The strains
NW_002477249, NW_002477237) of the reference strain A. flavus NRRL M1-8095, M2-8095, M3-8095, M4-8095, M7-8095, were totally in-
3357, to amplify and sequence the whole gene. After optimization of hibited by 500 mg/L of isopyrazam, with EC50 value included in the
PCR conditions, the amplification of SdhB, SdhC and SdhD genes with range 5–10 mg/L for the strains 8095-M1, and 10–50 mg/L for the
the specific primers designed in this study (Table 1) gave PCR products strains M2-8095, M3-8095, M4-8095, M7-8095 (Table 2). The strains
of the expected size (1384, 1096, 994 bp, respectively). The PCR am- M5-8095 and M6-8095 were inhibited by 100 mg/L with EC50 values
plicons were then purified, sequenced and assembled to obtain the included in the range10–50 mg/L.
complete sequence of the SdhB, SdhC, and SdhD genes. As well as boscalid, on PDA amended with 0.01 mg/L of iso-
By comparison with the mRNA sequence of A. flavus NRRL 3357, the pyrazam, all resistant mutants appeared yellow coloured.

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

Fig. 1. In vitro response of mutant strains to boscalid.

Inhibition of mycelial growth (a) and conidial germination (b), expressed in percentage, of Aspergillus flavus wild type strains (WT) and SDHI resistant genotypes
(sdhB-H249Y, sdhC-G91R), caused by boscalid. Mycelial growth was tested on potato dextrose agar amended with different concentration of boscalid, expressed in
mg/L, after 7 days incubation at 25 °C. Conidial germination was tested on water agar amended with different concentrations of boscalid, expressed in mg/L, after
48 h of incubation at 25 °C.

3.2.2. Conidial germination inhibition produce AFBs, while a different behaviour was observed for boscalid
In conidial germination test, wild type strains were totally inhibited resistant mutants. The strains with H249Y genotype (M1-8095, M2-
by 0.1 mg/L (ITEM 8111) or 0.5 mg/L (ITEM 8095, ITEM 8115) of 8095, M4-8095, M5-8095, M7-8095) produced high level of AFBs on
boscalid, as reported in Table 2. All resistant mutant carrying the PDA amended with 0.5 mg/L of boscalid, with a mean value of
H249Y amino-acid change in the SdhB gene were highly resistant, with 3570 μg/kg. These values ranged from 2915 to 5075 μg/kg, showing an
MIC and EC50 values higher than 1000 mg/L (mean inhibition value at increase from 3 to 10 times greater than the production on PDA without
highest concentration 11.2 ± 3.6%). The two strains M3-8095 and boscalid. Values near to production on PDA (from 732 to 1099 μg/kg)
M6-8095 showed MIC and EC50 values higher than 1000 mg/L were observed in presence of 1 μg/kg of boscalid, except for the strains
(Table 2), however at the highest concentration were more inhibited M4-8095 and M7-8095. Decreasing mean values of AFBs, about 300,
(mean value 34.3 ± 2.1%) than other resistant strains, as observed on 150 and 60 μg/kg, were detected with 5, 10 and 50 mg/L of boscalid,
mycelial growth test (Fig. 1b). respectively. An increment of AFBs production (mean value of 290 μg/
With regard to isopyrazam (Table 2 and Fig. 2b) all sensitive strains kg) was detected again with 100 μg/kg of boscalid. The strains carrying
were inhibited by 5 mg/L of molecule, showing EC50 value included in out the G91R genotype (M3-8095 and M6-8095) produced about
the range 1–5 mg/L. All resistant strains were totally inhibited at 700 μg/kg of AFBs on PDA, about 1300 μg/kg with 0.5 mg/L of boscalid
100 mg/L with EC50 values ranging between 1 and 50 mg/L. and about 240 μg/kg with 1 mg/L. At boscalid concentrations higher
than 1 μg/kg they didn't produce AFBs.
On PDA amended with isopyrazam (Fig. 4b), all the boscalid re-
3.3. Aflatoxin production of Sdh boscalid resistant mutants
sistant strains produced AFBs level comparable with control (only PDA,
about 700 μg/kg) when grown with 0.005 mg/L of fungicide (about
Aspergillus flavus boscalid resistant mutants grown on PDA showed
600 μg/kg), very high level (up to 6000 μg/kg, with a mean of about
AFBs production ranging from 366 to 1091 μg/kg, with a mean of
4000 μg/kg) with 0.01 mg/L of isopyrazam, including wild-type ITEM
705 μg/kg, compared to 897 μg/kg of AFBs produced by the wild-type
8095, and about 1000 μg/kg with 0.05 mg/L of isopyrazam con-
ITEM 8095.
centration. The strain ITEM 8095 did not produce AFBs with
On PDA amended with boscalid (Fig. 4a), ITEM 8095 did not

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

Fig. 2. In vitro response of mutant strains to isopyrazam.

Inhibition of mycelial growth (a) and conidial germination (b), expressed in percentage, of Aspergillus flavus wild type strains (WT) and SDHI resistant genotypes
(sdhB-H249Y, sdhC-G91R), caused by isopyrazam. Mycelial growth was tested on potato dextrose agar amended with different concentration of isopyrazam, ex-
pressed in mg/L, after 7 days incubation at 25 °C. Conidial germination was tested on water agar amended with different concentrations of isopyrazam, expressed in
mg/L, after 48 h of incubation at 25 °C.

isopyrazam concentrations higher than 0.01 mg/L. With increasing these three isopyrazam concentrations. Mutant strains with H249Y
concentrations of isopyrazam (0.1, 0.5, 1 mg/L) AFBs production of all genotype did not produce AFBs at isopyrazam concentrations higher
resistant strains decreased (mean values of about 650, 57, 34 μg/kg, than 1 mg/L; mutant strains with G91R genotype didn't produce AFBs
respectively), except M3-8095 that produced about 350 μg/kg with at isopyrazam concentrations higher than 5 mg/L.

Table 2
Boscalid and isopyrazam concentration causing 50% reduction (EC50) and minimal inhibitory concentration (MIC) values, expressed as mg/L, evaluated on
Aspergillus flavus mycelial growth and conidial germination. The molecular genotype referring to Sdh genes is also reported for A. flavus strains used as wild-type (WT)
and for mutant strains resistant to boscalid (SdhB-H249Y, SdhC-G91R).
Strain Genotype Boscalid Isopyrazam

Mycelial growth Conidial germination Mycelial growth Conidial germination

MIC EC50 MIC EC50 MIC EC50 MIC EC50

ITEM 8095 WT 50 0.05–0.1 0.5 0.01–0.05 50 0.1–0.5 10 1–5

ITEM 8111 WT 50 0.1–0.5 0.1 0.005–0.01 50 0.1–0.5 5 1–5
ITEM 8115 WT 50 0.05–0.1 0.5 0.05–0.1 50 0.1–0.5 5 1–5
M1-8095 SdhB-H249Y > 1000 > 1000 > 1000 > 1000 500 5–10 100 10–50
M2-8095 SdhB-H249Y > 1000 > 1000 > 1000 > 1000 500 10–50 100 1–5
M4-8095 SdhB-H249Y > 1000 > 1000 > 1000 > 1000 500 10–50 100 10–50
M5-8095 SdhB-H249Y > 1000 > 1000 > 1000 > 1000 100 10–50 100 0.5–1
M7-8095 SdhB-H249Y > 1000 > 1000 > 1000 > 1000 500 10–50 100 5–10
M3-8095 SdhC-G91R > 1000 > 1000 > 1000 > 1000 500 10–50 100 10–50
M6-8095 SdhC-G91R > 1000 > 1000 > 1000 > 1000 100 10–50 100 5–10

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

Fig. 3. Colony pigmentation of mutant strains at different boscalid concentrations.

Colony pigmentation of mutant strains grown on Potato Dextrose medium (PDA) amended with different concentrations (0.5, 1 and 5 mg/L) of boscalid.

3.4. Statistical significance of AFs production at sub-lethal doses of SDHIs registered in reducing A. flavus contamination of maize. The group of
SDHIs fungicides is recognized to have a medium-high risk of re-
After running post-hoc Tukey's HSD, the mean of AFs amount at sistance. No information are available on the ability of A. flavus strains
0.5 mg/L boscalid concentration was significantly different from all the to acquire resistance towards boscalid.
other boscalid concentrations, with p value = 0.01. Likewise, the mean We selected 7 A. flavus laboratory mutants resistant to boscalid,
of AFs at 0.01 mg/L of isopyrazam was statistically different from all with a frequency of 1.4 × 10−10 conidia. All have shown high re-
the other isopyrazam concentrations, with p value = 0.01. sistance to boscalid and moderate resistance to isopyrazam. Aspergillus
In addition, significant correlation values (p < 0.01) were found flavus has a great capability to colonize several agri-food products in
between AFs production and colonies pigmentation of A. flavus strains pre- and post-harvest conditions by producing a large amount of con-
exposed to different concentrations of boscalid (r = 0.83) and iso- idia. Thus, since A. flavus has a high capability to acquire fast resistance
pyrazam (r = 0.86), respectively. to SDHIs, we suppose that repeated treatments in the field with these
compounds could reduce their effectiveness.
4. Discussion Nowadays, almost all fungicides control fungal development by
interfering with specific metabolic pathways. As a consequence, the
To date, SDHI are not registered to control A. flavus development. resistance is associated to polymorphisms in specific gene target sites.
However, the two SDHI fungicides boscalid and isopyrazam showed to In many phytopathogenic fungi, several mutations involved in SDHI-
be effective in the reduction of A. flavus growth in vitro (Masiello et al., resistance are known within the SdhB, SdhC and SdhD genes, encoding
2019). Moreover, in field conditions, a chemical treatment with bos- the iron‑sulfur protein and the two anchor proteins of the succinate
calid at flowering time was reported to be able to reduce A. flavus dehydrogenase complex, respectively (Avenot and Michailides, 2010;
contamination of maize kernels (Masiello et al., 2019). Thus, boscalid Leroux et al., 2010). Due to differences in length of the Sdh genes in
effectiveness both in vitro and in field, the specific mode of action and different fungal species, homolog positions of the mutations do not
the activity against a broad spectrum of fungi that can simultaneously necessarily have the same amino acid number when compared across
co-occur on crops, make it this compound a good candidate to be different species. The amino acid 267 of SdhB of M. graminicola is

Fig. 4. Aflatoxin B production at different concentrations of boscalid and isopyrazam.

Aflatoxin B production, expressed in μg/kg, of Aspergillus flavus wild type strain (ITEM 8095) and SDHI mutant strains (M1-8095, M2-8095, M3-8095, M4-8095, M5-
8095, M6-8095, M7-8095) grown on potato dextrose agar amended with different concentrations of boscalid (a) and isopyrazam (b), expressed in mg/L.

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

homologous to position 249 in A. oryzae, to position 257 in U. maydis, with moderate resistance (MIC in colony growth test ranging between
to position 272 in P. expansum and B. cinerea, and to position 277 in D. 100 and 500 mg/L). Our results agree with several studies reporting
bryoniae, Alternaria alternata and Pyrenophora teres (Malandrakis et al., positive cross resistance between boscalid and isopyrazam in other
2017; Sierotzki and Scalliet, 2013). In addition, the replace of Histidine fungal species, such as A. alternata, B. cinerea, M. graminicola and P.
with Tyrosine in the SdhB subunit at 267 or 272 or 277 homologous expansum (Avenot et al., 2008b; Malandrakis et al., 2017; Skinner et al.,
positions is the most frequent mutation occurring in different fungal 1998; Veloukas et al., 2013).
species (Sierotzki and Scalliet, 2013). Since A. flavus is the most important aflatoxigenic species, we
In A. flavus, sdh genes associated to resistance were never in- evaluated also the capability of resistant laboratory mutant strains to
vestigated. In order to identify the genetic sites responsible for the re- produce AFBs. Indeed, according to the Scientific Committee on Plants
sistance to boscalid in A. flavus species, we characterized for the first of the European Commission (1999), fungicides can influence myco-
time sdh genes in sensitive and resistant A. flavus strains. Specific pri- toxin production mostly as an indirect effect, due to the inhibition of
mers to amplify the whole sdh genes in A. flavus have been designed. fungal development. However, some studies assessed a direct effect of
The analyses of nucleotide and amino acid sequences, demonstrated fungicides on mycotoxin production in vitro, as reported in Fusarium
that five boscalid-resistant mutant strains differed from the wild-type culmorum for deoxynivalenol (DON) production (Siranidou and
strain by a single point mutation leading to an amino-acid substitution Buchenauer, 2001), A. flavus for AFBs (Sadhasivam et al., 2019), and
in the third cysteine-rich regions of SdhB protein. The remaining two Aspergillus carbonarius for ochratoxin A (OTA) (Malandrakis et al.,
mutants, sharing the same SdhB sequence of wild-type, showed a single 2013). On the contrary, in other studies, production of T-2 toxin and
point mutation in SdhC gene. In particular, in the present work, for the diacetoxyscirpenol by Fusarium sporotrichioides, and AFs by A. flavus
first time, mutation at the codon 249 of the SdhB gene, leading to were enhanced under the pressure of fungicides, despite a reduced
substitution of Histidine by Tyrosine (H249Y) and mutation at the fungal growth (Badii and Moss, 1988; Moss and Frank, 1985). Fur-
codon 91 of the SdhC gene, leading to substitution of Arginine by thermore, experiments using fungicides to control toxigenic fungi, have
Glycine (G91R), both were shown to be associated to high resistance to shown that laboratory resistant mutant strains could produce higher
boscalid coupled with moderate resistance to isopyrazam, in A. flavus. amount of mycotoxins than wild-type. This is the case of P. expansum
The design of specific primer pairs for A. flavus able to amplify the strains resistant to boscalid, with respect to patulin (Malandrakis et al.,
whole Sdh subunits and the optimization of PCR conditions, allow 2017), A. parasiticus resistant to fludioxonil for the production of AFBs
screening, within a set of A. flavus strains, mutants with resistance to (Markoglou et al., 2008), and A. carbonarius strains resistant to flu-
boscalid. This provides a useful molecular tool for the identification dioxonil for the production of OTA (Markoglou et al., 2011).
either of the point mutations reported in this study or new allelic var- In our study, all the A. flavus laboratory mutant strains produced
iations responsible of different response to SDHI in A. flavus popula- AFBs in presence of boscalid and isopyrazam. Differences were also
tions. observed between the two resistant genotypes, since H249Y strains
As widely observed in other fungal species, different point muta- were able to produce AFBs up to a concentration of boscalid (100 mg/L)
tions, occurring in the same gene region, can confer different resistance 10-fold higher than G91R strains. On the contrary, on isopyrazam,
levels. Indeed, we identified two different point mutations (genotypes G91R genotype strains produced AFBs at a higher concentration of
H249Y and G91R) that reflect differences in their responses to boscalid isopyrazam than H249Y genotype. Therefore, we established a strong
for mycelial growth and conidial germination inhibition. Strains pro- correlation between application of fungicides and AFBs synthesis.
vided of H249Y genotype showed high resistance, growing also at the Indeed, some authors have demonstrated a direct action of fungicides
highest boscalid concentration. They were able to grow and to germi- towards mycotoxin biosynthetic genes. In particular, fungicides appli-
nate on boscalid, showing inhibition up to 25% for mycelial growth and cation in Fusarium genus enhanced the expression of the tri5 gene, a key
up to 11% for conidial germination. Strains provided of genotype G91R, gene of the trichothecene biosynthetic pathway (Doohan et al., 1999).
showed higher inhibition values (38% and 35% for mycelial growth and Also, Covarelli et al. (2004) reported that trifloxystrobin reduced sig-
conidial germination, respectively) than genotype H249Y. This suggests nificantly DON production in F. culmorum, influencing the expression of
that mutation in SdhB sub-unit is more specific in conferring resistance tri5 and tri6 genes. However, a deeper understanding of the molecular
than mutation in SdhC. Moreover, while mycelial growth differs only mechanisms that are activated in promoting toxicogenesis under the
slightly between the genotypes, boscalid was ineffective to inhibit pressure of fungicides would be worthwhile.
conidial germination and germ tube elongation of strains with H249Y Specific investigations focused on this topic are in progress on our
genotype at all the concentrations, while strains with G91R genotype strains to better explain the significant, if not dramatic, increase of AFBs
were slightly inhibited only at the two highest concentrations. production observed at specific sub-lethal fungicide concentrations of
However, our results on wild-type A. flavus strains showed that both fungicides tested (0.5 mg/L of boscalid and 0.01 mg/L of iso-
boscalid inhibited conidial germination more than mycelial growth, pyrazam). Similar findings, showing increasing mycotoxin production
that agrees with previous observations in other fungal species, such as at sub-lethal doses of fungicides, were also reported in other studies.
Botrytis cinerea (De Miccolis Angelini et al., 2014). On the other hand, Sub lethal dose of prochloraz in mixture with tebuconazole triggered a
by comparing wild-type and resistant mutants, boscalid showed to be higher AFBs production by A. flavus (Mateo et al., 2017), while in-
ineffective against all the resistant mutants, both in mycelia growth and creased production of DON and HT-2 toxin by F. graminearum, F. cul-
in conidial germination. morum and F. sporotrichioides (D'Mello et al., 1998, 2000; Magan et al.,
Laboratory mutants or field strains provided of high resistance to 2002), following exposure to sub-lethal doses of fungicides, have also
boscalid have been reported for several fungal species, including A. been demonstrated. Likewise, also several natural products at sub-lethal
alternata, Alternaria solani, B. cinerea, A. oryzae, M. graminicola, and P. concentrations were reported to stimulate the production of mycotoxins
expansum (Malandrakis et al., 2017; Sierotzki and Scalliet, 2013). by Fusarium and Aspergillus species, although they reduced the colony
However, this is the first report on the selection of laboratory mutant growth (Hope et al., 2005; Morcia et al., 2017; Nerilo et al., 2016).
strains showing high boscalid resistance in A. flavus. Usually, when a Despite the studies carried out, the mechanisms by which the fungicides
fungal pathogen develops resistance to a fungicide, it becomes resistant stimulate toxin production are not clear yet. Fungicides can act as a
to all the others having the same mode of action (positive cross re- stress factor stimulating synthesis of mycotoxins as a fungal defence
sistance), although cross-resistance towards SDHI fungicides in several response, as reported by Audenaert et al. (2010), that proved an in-
fungal species appeared to be limited to specific boscalid-resistant creased production of DON by F. graminearum, in vitro and in planta,
genotypes (De Miccolis Angelini et al., 2014). Likely, all our boscalid when exposed to sub-lethal doses of prothioconazole, hypothesizing
resistant mutants have shown resistance also to isopyrazam, although that the biosynthesis was stimulated by an over production of hydrogen

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M. Masiello, et al. International Journal of Food Microbiology 334 (2020) 108799

peroxide (H2O2), induced by the fungicide. The use of sub-lethal con- Antonio Moretti: Funding acquisition, Project administration,
centrations of azoles, which class prothioconazole belongs, confirmed Resources, Supervision, Writing - review & editing.
Audenaert et al. (2010) finding at molecular level, as Ochiai et al.
(2007) and Kulik et al. (2012) reported that these compounds activate Declaration of competing interest
transcription of tri genes in F. graminearum.
Noteworthy, when we exposed A. flavus at some specific sub-lethal None.
concentrations of boscalid and isopyrazam, we observed both a strong
depigmentation of SDHI resistant strain colonies, that appeared light Acknowledgements
yellow, and a correlated peak in aflatoxin production. On the contrary,
no significant differences in means of aflatoxin production between We thank Dr. Giuseppe Cozzi for his valuable technical assistance in
wild type strain and mutants was observed when depigmentation did statistical analyses.
not occur.
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